Autophagy is a highly regulated, critical process that cells rely upon for stress response, differentiation, tissue remodeling, and apoptosis. Autophagy is remarkably well-conserved in eukaryotes ranging from yeast to humans, and some of the core autophagy (ATG) proteins can be found in early-branching eukaryotes such as the pathogenic parasite Toxoplasma gondii. Interestingly, Toxoplasma exhibits some features of canonical autophagy upon amino acid starvation, such as redistribution and lipidation of autophagy protein ATG8 (TgATG8); however, many core components of the autophagy machinery are missing or highly divergent, suggesting that autophagy is streamlined in this parasite or its ATG proteins have unique functions. The observation that TgATG8 associates with the essential plastid-like organelle known as the apicoplast in a lipidation- dependent manner supports the idea that ATG proteins possess novel functions in Toxoplasma. TgATG8 and other TgATGs are essential for parasite survival, which further warrants their importance for study, but prohibits the generation of knockout parasites as a tool to understand their biological role. Thus, alternative approaches will be required to interrogate this important class of proteins in Toxoplasma. We have recently found that lysine 23 (K23) of TgATG8 is acetylated and hypothesize that the study of post-translational modifications (PTMs) will yield vital new insights into TgATG proteins. As proof of principle, we mutated K23 to a nonacetylatable amino acid and surprisingly found spontaneous encystation occurring in mutant parasites. Thus, we have discovered a novel role for TgATG8 in the conversion of replicating parasites to their latent stage. We propose to elucidate the mechanism underlying this striking phenotype, thereby filling a knowledge gap linking autophagy homologues and cyst conversion. To begin deciphering the functionality of TgATG8, we will determine the timing of its acetylation and monitor autophagic flux, TgATG8 localization, and TgATG8 protein interactions in K23 mutant parasites. Furthermore, we will determine the role of TgATG8 acetylation in parasite development and virulence through in vivo experiments. To establish a new understanding of the role of TgATG8, we will also determine the localization and PTM occurring on the core TgATG8- conjugation complex homologues TgATG3, TgATG4, TgATG5, and TgATG7 in normal versus autophagic conditions. These studies will provide a wealth of new data for the research community, exposing new avenues to investigate canonical and noncanonical roles of the TgATG8-conjugation system, which promises to lead to new targets for treatment of toxoplasmosis and related parasitic diseases.